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Notable among the original or early acquisitions of the Pathological Collections are the exsiccati from J.B. Ellis, G.L. Rabenhorst, and H.W. Ravenel. Other early collections are from T.J. Burrell, G.W. Clinton, B.D. Halsted, E.W.D. Holway, M.E. Jones, W.A. Kellerman, and A.B. Langlois. Specimens were submitted from the U.S. Exploring Expedition under Commodore Wilkes, 1838-42, and also from the U.S. North Pacific Exploring Expedition under Commanders Ringgold and Rogers, 1853-56. The earliest American collections of fungi were those by L.D von Schweinitz. Many of the Schweinitz collections and those by M.A. Curtis and other early American mycologists are in the U.S. National Fungus Collections.

Thousands of collections also were contributed by mycologists and plant pathologists of the U.S. Department of Agriculture. Some of the most extensive are from G.G. Hedgecock, Jenkins, Shear, Stevenson, and Weir. In 1924 the valuable G. Bresadola herbarium from Italy was purchased for the U.S. National Fungus Collections and the E.A. Rau herbarium, including types, was acquired as a gift. The C.G. Lloyd herbarium was obtained in 1927. With more than 59,000 collections, it may be the largest fungus herbarium ever assembled by one person. Weir's herbarium of wood-decay fungi, acquired in 1928, also numbered many thousands of collections.

Other major acquisitions include the P.C. Standley collection from Honduras and Costa Rica; L.W. Nuttall fungi from West Virginia; remainders of the J.B. Ellis herbarium; the Sbarbaro collections from Europe, New York and Colorado collections by Shear collections by E.A. Burt, specimens from the former Division of Cereal Crops and Disease collections contributed by the Horticultural Crops and Diseases Division (USDA), the herbarium of R. Ciferri from the Dominican Republic; O. A. Reinking's Philippine collections; the G.L. Zundel Smut herbarium; E.R. Bethel's herbarium of Colorado fungi; the W.H. Long collections of rust fungi and gasteromycetes; C.E. Chardon's herbarium of parasitic fungi from the West Indies and Central and South America; Japanese fungi from N. Hiratsuka; type specimens of P.A. Karsten's European wood-decay fungi; remainders of the Chinese National Herbarium, the J.H. Faull rust fungi; Elizabeth B. Morse's mushrooms; E.F. Guba's collections of Pestalotia; and the entire mycological herbaria of Goucher College, Stanford University, the Missouri Botanical Garden, and the Brooklyn Botanic Garden, including the large personal herbarium of F. Bubak and the collections of D. Griffiths and G.M. Reed. In 1953 the fungus herbarium of the New Haven, Connecticut Forest Service Field Station was transferred to the U.S. National Fungus Collections.

A relatively recent acquisition is the herbarium of the Connecticut Agricultural Experiment Station at New Haven. It includes the smut fungi of G.P. Clinton. Another addition was the myxomycetes herbaria of T.H. Macbride and G.W. Martin from the State University of Iowa, as well as myxomycetes from C.J. Alexopoulos, T. Brooks, and D. Reynolds. As a result of these acquisitions, the myxomycete section in the U.S. National Fungus Collections is now among the most notable in the world. The same is true of the polypore and rust collections, as well as several other outstanding sections of the herbarium. Collections also have been received from such well-known scientists as R. Allen, J.P. Anderson, R.K. Beattie, W.W Calkins, G.W. Carver, F.S. Earle, F.D. Fromme, F.D. Hall, J.R. Hansbrough, A.G. Johnson, C.L. Lefebrve, A. Liberta, W.H. Long, W.A. Orton, J. Rick, W.H. Snell, A.B. Seymour, E.F. Smith, F.L. Stevens, N.E. Stevens, W.T. Swingle, B.C. Tharp, S.M. Tracy, M.B. Waite, H.J. Webber, F.H. Wellman, F.A. Wolf, and many others. S. Ahmad in Pakistan and K.S. Thind in India have contributed numerous collections. Also, a policy was established by Patterson and followed by her successors for the acquisition of all sets of fungi exsiccati as they became available by purchase or exchange.

As the herbarium grew, a reference literature collection and extensive data files were developed. The library was built up principally as the personal collection of Stevenson who donated it to the Smithsonian Institution. In 1976 an understanding between the Smithsonian Institution and the Agricultural Research Service provided for permanent maintenance by the research staff of the U.S. National Fungus Collections.

In November 2002 the Mycological Herbarium of the Pennsylvania State University (PACMA) was moved from Mont Alto, Pennsylvania, to the U.S. National Fungus Collections. The PACMA herbarium, which consists of about 67,000 specimens including about 1,000 type specimens, is being incorporated into BPI starting with the type specimens, Uredinales (rusts), Ustilaginales (smuts), Deuteromycetes (imperfect fungi), and Ascomycetes. All specimen data will eventually be made available to scientists as part of the BPI specimen database.